The invention relates to regulators of fungal gene expression and their use in commercial and medical applications. More particularly, the invention relates to regulators of fungal genes involved in production of enzymes, secondary metabolites and other useful products, as well as to regulators of genes involved in fungal invasion.
Fungi are among the most common natural sources of useful substances for commercial and medical applications. Many of these useful substances are secondary metabolites.
Secondary metabolite production by various fungi has been an extremely important source of a variety of therapeutically significant pharmaceuticals. xcex2-lactam antibacterials such as penicillin and cephalosporin are produced by Penicillium chrysogenum and Acremonium chrysogenum, respectively, and these compounds are by far the most frequently used antibacterials (reviewed in Luengo and Penalva, Prog. Ind. Microbiol. 29: 603-38 (1994); Jensen and Demain, Biotechnology 28: 239-68 (1995); Brakhage, Microbiol. Mol. Biol. Rev. 62: 547-85 (1998)). Cyclosporin A, a member of a class of cyclic undecapeptides, is produced by Tolypocladium inflatum. Cyclosporin A dramatically reduces morbidity and increases survival rates in transplant patients (Borel, Prog. Allergy 38: 9-18 (1986)). In addition, several fungal secondary metabolites are cholesterol-lowering drugs, including lovastatin that is made by Aspergillus terreus and several other fungi (Alberts et al., Proc. Natl. Acad. Sci. USA 77: 3957-3961 (1980)). These and many other fungal secondary metabolites have contributed greatly to health care throughout the world (see Demain, Ciba Found Symp 171: 3-16 (1992); Bentley, Crit. Rev. Biotechnol. 19: 1-40 (1999)).
Unfortunately, many challenges are encountered between the detection of a secondary metabolite activity and production of significant quantities of pure drug. Thus, efforts have been made to improve the production of secondary metabolites by fungi. Recently, strains have been improved by manipulating genes encoding the biosynthetic enzymes that catalyze the reactions required for production of secondary metabolites. Penalva et al., Trends Biotechnol. 16: 483-489 (1998) discloses that production strains of P. chrysogenum have increased copy number of the penicillin synthesis structural genes. Other studies have modulated expression of other biosynthetic enzyme-encoding genes, thereby affecting overall metabolism in the fungus. Mingo et al., J. Biol. Chem. 21: 14545-14550 (1999), demonstrate that disruption of the gene encoding phacA, an enzyme in A. nidulans that catalyzes phenylacetate 2-hydroxylation, leads to increased penicillin production, probably by elimination of competition for the substrate phenylacetate. Similarly, disruption of the gene encoding aminoadipate reductase in P. chrysogenum increased penicillin production, presumably by eliminating competition for the substrate alpha-aminoadipate (Casquiero et al., J. Bacteriol. 181: 1181-1188 (1999)).
Thus, genetic manipulation holds promise for improving production of secondary metabolites. Genetic manipulation to increase the activity of biosynthetic enzymes for secondary metabolite production or to decrease the activity of competing biosynthetic pathways has proven effective for improving production. Maximum benefit might be achieved by combining several strategies of manipulation. For example, modulating the expression of genes that regulate the biosynthetic enzyme-encoding genes or altering concentrations of metabolic precursors might improve production. In addition, genetic manipulation could be used to impact upon the challenges that are encountered in the fermenter run or downstream processing (e.g. energy cost, specific production of desired metabolite, maximal recovery of metabolite, cost of processing waste from fermentations). There is, therefore, a need for regulator genes that can improve secondary metabolite production in a fungus.
Enzymes are another commercially important fungal product. Recently, efforts have been made to improve fungal enzyme production through genetic manipulation. Noel et al., Molecular Microbiology 27: 131-142 (1998), teaches that xlnR, a ZBC protein, induces expression of xylanolytic extracellular enzymes in Aspergillus niger. Hasper et al., Molecular Microbiology 36: 193-200 (2000) teaches that xlnR also regulates D-xylose reductase gene expression in Aspergillus niger. Given the many useful enzymes produced by fungi, there remains a need for regulator genes that can improve the production of these enzymes.
There is also a need for regulator identifying genes relevant to fungal invasion. Fungal invasion is required for fungal pathogenesis and its regulation is likely related to secondary metabolite and enzyme production. Fungal infections have become a serious health concern, especially in immunocompromised patients. Ha and White, Antimicrobial Agents and Chemotherapy 43: 763-768 (1999) teach that candidiasis, which is caused by the pathogenic yeast Candida albicans, is the most frequent fungal infection associated with AIDS and other immunocompromised states. Weig et al., Trends in Microbiology 6: 468-470 (1998) discloses that the frequency of Candida infections has increased in recent years and has been accompanied by a significant rise in morbidity and mortality. Many of these infections take place in the hospital setting. Baillie and Douglas, Methods in Enzymology 310: 644-656 (1999) teach that a majority of nosocomial septicemias caused by Candida species derive from biofilm formation on catheters and shunts. Little is known about the genes necessary for invasion or biofilm formation. There is, therefore, a need for the identification of new fungal invasion regulatory genes to act as targets for the development of antifungal drugs.
The invention provides novel fungal regulator genes and methods for using regulator genes in commercial and medical applications.
In a first aspect, the invention provides novel isolated or recombinant genes that have been demonstrated to encode proteins that regulate fungal genes that are involved in secondary metabolite production, enzyme production, or fungal invasion. In certain preferred embodiments, the invention further provides homologs of such genes. These genes and their homologs are useful for improving secondary metabolite or enzyme production, or as targets for discovering new antifungal drugs.
In a second aspect, the invention provides isolated or recombinant nucleic acids that are specifically complementary to genes that have been demonstrated to encode proteins that regulate fungal genes that are involved in secondary metabolite production, enzyme production, or fungal invasion.
In a third aspect, the invention provides purified proteins that have been demonstrated to regulate fungal genes that are involved in secondary metabolite production, enzyme production, or fungal invasion. In certain embodiments, the invention further provides homologs of such proteins.
In a fourth aspect, the invention provides novel binding agents that specifically bind to proteins that have been demonstrated to regulate fungal genes that are involved in secondary metabolite production, enzyme production, or fungal invasion.
In a fifth aspect, the invention provides novel recombinant genes that are direct or indirect regulators of expression of FLO11, a fungal gene that is required for fungal invasion and whose expression is believed to be regulated by factors that also modulate secondary metabolite production. In certain embodiments, the invention further provides homologs of such genes. These genes are useful as targets for the development of antifungal drugs, and are expected to be useful for improving the production of secondary metabolites or fungal enzymes.
In a sixth aspect, the invention provides novel recombinant genes that are direct or indirect regulators of expression of lovF, a fungal gene involved in the production of the secondary metabolite lovastatin. In certain embodiments, the invention further provides homologs of such genes. These genes are expected to be useful for improving the production of secondary metabolites or fungal enzymes.
In a seventh aspect, the invention provides novel recombinant genes that are direct or indirect regulators of expression of lovE, a fungal gene involved in the production of the secondary metabolite lovastatin. In certain embodiments, the invention further provides homologs of such genes. These genes are expected to be useful for improving the production of secondary metabolites or fungal enzymes.
In an eighth aspect, the invention provides novel recombinant genes that are direct or indirect regulators of expression of acvA, a fungal gene involved in the production of the secondary metabolite penicillin. In certain embodiments, the invention further provides homologs of such genes. These genes are expected to be useful for improving the production of secondary metabolites or fungal enzymes.
In a ninth aspect, the invention provides methods for modulating production of a secondary metabolite or enzyme, the method comprising expressing in the fungus a novel fungal regulator gene.
In a tenth aspect, the invention provides novel chimeric fungal regulator genes.
In an eleventh aspect, the invention provides methods for modulating production of a secondary metabolite or extracellular enzyme, the method comprising expressing in the fungus a novel chimeric fungal regulator gene.